Antibiotics aclacinomycins A and B

ABSTRACT

New antitumor agents named aclacinomycins A and B, which are anthracycline glycosides and inhibit the growth of various microorganisms e.g., Staphylococcus aureus, Micrococcus flavus, Corynebacterium bovis and inhibit the growth of animal tumors such as leukemia L1210 and P388 and lymphoma 6C3HED in mice and hepatomas in rats are produced by the fermentation of a microorganism belonging to the genus Streptomyces which has been designated Streptomyces galilaeus (MA144-M1 and A.T.C.C. 31133); they are recovered from the broth by conventional methods for recovering antibiotics.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to new anthracycline glycoside antitumorantibiotics and to their production. More particularly, it relates tonew antitumor antibiotic substances designated aclacinomycins A and Band to processes for the preparation thereof by the fermentation of astrain Streptomyces galilaeus (e.g., MA144-M1), to methods for theirrecovery and purification, and to their application as cancerchemotherapeutic agents.

2. Description of the Prior Art

A number of anthracycline glycosides have been found in the culturedbroth of Streptomyces. Among them, daunomycin and adriamycin areparticularly being watched with keen interest in the field of cancerchemotherapy and have already been applied clinically for human cancers.In the continuation of the study of antitumor antibiotics, the presentinventors discovered new compounds and after characterization andpurification based on their physico-chemical properties, they confirmedthat these antibiotics now named aclacinomycin A and B are new compoundswhich show low cardiotoxicity and potent antitumor activity in variousanimal tumors, and they established processes and methods for theirproduction and isolation.

Farmitalia's U.S. patent on adriamycin (B-106FI; 14-hydroxy-daunomycin;INN is Doxorubicin) is U.S. Pat. No. 3,590,028 claiming the product bystructure and disclosing its direct fermentation by S. peuceticus var.caesius. Farmitalia also issued U.S. Pat. No. 3,803,124 on chemicalconversion of daunomycin to adriamycin; for direct fermentation ofdaunomycin (as antibiotic FI 1762) by S. peuceticus see U.K. Pat. No.1,003,383.

Farmitalia's daunomycin (U.S. Pat. No. 1,003,383) may be the same asRhone-Poulenc's 13057 R.P. (formerly rubidomycin and now Daunoribicin(U.K. Pat. Nos. 985,598; 1,188,262; 1,241,750 and see U.S. Pat. No.3,616,242) and is "probably" identical with Ciba's danubomycin (U.S.Pat. No. 3,092,550; U.K. Pat. No. 901,830). See also U.S. Pat. No.3,686,163 on dihydrodaunomycin.

Cinerubin A and cinerubin B are disclosed in U.K. Pat. No. 846,130 andsee also U.S. Pat. No. 3,864,480 and Keller-Schierlein et al.,Antimicrobial Agents and Chemotherapy, page 68 (1970) and ChemicalAbstracts, 54, 1466i (1960).

For further illustrative and summary disclosures of anthracyclineantibiotics see Index of Antibiotics from Actinomycetes, Hamao Umezawa,Editor-in-chief, University Park Press, State College, Pennsylvania,U.S.A. (1967) as follows:

    ______________________________________                                        Antibiotic        Page Number                                                 ______________________________________                                        Aklavin           111                                                         Cinerubin A       220                                                         Cinerubin B       221                                                         Danubomycin       242                                                         Daunomycin        243                                                         Pyrromycin        542                                                         Rhodomycin A,B    561                                                         Rubidomycin       574                                                         ______________________________________                                    

The textbook Antibiotics, Volume 1, Mechanism of Action, edited by DavidGottlieb and Paul D. Shaw, Springer-Verlag New York, Inc., N.Y., N.Y.(1967) at pages 190-210 contains a review by A. DiMarco entitledDaunomycin and Related Antibiotics.

Information Bulletin, No. 10, International Center of Information ofAntibiotics, in collaboration with WHO, December, 1972, Belgium, reviewsanthracyclines and their derivatives

For a description of Streptomyces galilaeus see Arch. fur Mikrobiol.,31, 356 (1958) and International Journal of Systematic Bacteriology, 22,298 (1972).

This specification on page 37 refers to disclosures by K. Eckardt et al.on galirubins; see Chemical Abstracts 64, 3896g and 67, 90573z.

SUMMARY OF THE INVENTION

There are provided by the present invention the antitumor agentsaclacinomycins A and B. These substances are produced by cultivating anaclacinomycin-producing strain of Streptomyces galilaeus in an aqueouscarbohydrate solution containing organic nitrogenous nutrients undersubmerged aerobic conditions until a substantial quantity ofaclacinomycin A and B is formed in said solution. Aclacinomycin A and Bin cultured broths thus produced can be extracted and purified byconventional methods used for extraction and purification ofwater-insoluble antibiotics. This invention also embraces aclacinomycinsA and B as crude solids, as purified solids, as their salts and asDNA-complexes.

There is thus provided by the present invention the antitumor antibioticaclacinomycin A which

a. is effective in inhibiting the growth of ascites and solid forms ofEhrlich carcinoma, Sarcoma 180, lymphoma 6C3HED and leukemia L1210 andP388 in mice, hepatomas in rats and vaccina virus in HeLa cells;

b. is effective in preventing the growth of various Gram-positivebacteria and yeasts;

c. can be isolated as a yellow microcrystalline powder;

d. is soluble in organic solvents such as methanol, chloroform, ethanol,ethyl acetate, acetone and benzene, slightly soluble in water, butinsoluble in ether, hexane and petroleum ether;

e. has a melting point of 129° to 135° C., and is optically active with[α]_(D) ²⁴ = +29° (C: 1.0, CHCl₃);

f. exhibits ultraviolet and visible absorption maxima at 229.5, 258, 290and 434 nm in methanol; 229.5, 258, 290 and 434 nm in 0.1 NHCl-methanol; 239.5, 288, 314 and 523 nm in 0.1 N NaOH-methanol;

g. exhibits characteristic maxima in the infrared absorption spectrum at3300, 3080, 2940, 2860, 1740, 1640, 1620, 1540, 1460, 1450, 1415, 1385,1295, 1250, 1220, 1195, 1165, 1125, 1105, 1090, 1010, 960, 930, 890,840, 815, 760, 730, 580 and 450 cm.sup.⁻¹ ;

h. gives upon acid hydrolysis rhodosamine, 2-deoxyfucose, cinerulose A,1-deoxyprromycin and alkavinone;

i. is a weakly basic anthracycline glycoside having the empiricalformula of C₄₂ H₅₄ NO₁₅ ;

and the antitumor antibiotic aclacinomycin B which:

a. is effective in inhibiting the growth of leukemia L1210 and P388 inmice and vaccinia virus in HeLa cells;

b. is effective in inhibiting the growth of Gram-positive bacteria andyeasts;

c. can be isolated as a yellow microcrystalline powder;

d. is soluble in methanol, ethyl acetate, chloroform, ethanol, acetoneand benzene, slightly soluble in water, but insoluble in ether,cyclohexane, hexane and petroleum ether;

e. has a melting point of 135° to 145° C., and is optically active with[α]_(D) ²⁴ +3° (C: 1.0, CHCl₃);

f. exhibits ultraviolet and visible absorption maxima at 229.5, 257,5,290 and 433 nm in methanol, 229.5, 257.5, 290 and 433 nm in 0.1 NHCl-methanol, 238, 286 to 289, 315 and 525 nm in 0.1N NaOH-methanol;

g. exhibits characteristic maxima in the infrared absorption spectrum at3300, 3070, 2940, 2850, 2750, 1740, 1640, 1620, 1540, 1465, 1445, 1410,1380, 1290, 1245, 1215, 1160, 1120, 1095, 1050, 1010, 960, 920, 880,840, 820, 805, 750, 725, 700, 600, 580 and 450 cm.sup.⁻¹ ;

h. gives aklavinone, rhodosamine, 2-deoxyfucose, 1-deoxy-pyrromycin andthe same methylated disaccharide as cinerubin B on acid hydrolysis orpartial hydrolysis in methanol;

i. is a weakly basic anthracycline glycoside having the empiricalformula of C₄₂ H₅₂ NO₁₅.

There is further provided by the present invention the process forproducing the antitumor antibiotics aclacinomycins A and B whichcomprises culturing an aclacinomycin-producing strain of Streptomycesgalilaeus having the identifying characteristics of A.T.C.C. 31133 undersubmerged aerobic conditions in a nutrient medium containing carbonsources and nitrogenous nutrients until a substantial amount ofaclacinomycin A and B is produced by the said organism in the saidnutrient medium and preferably by culturing in a nutrient medium at atemperature in the range of 20° to 35° C. or better yet at a temperaturein the range of 25° to 30° C. with the pH from 5 to 8 and then, ifdesired, recovering the aclacinomycins from the culture medium by aprocess which includes at least one process selected from the groupconsisting of solvent extraction, solvent precipitation, concentration,gel filtration, counter current distribution, chelation with metal ionsand adsorption followed by elution from an ion exchange resin, adsorbentsiliceous earth material or synthetic adsorbent.

The processes of this invention include a process in which the solutioncontaining aclacinomycins is freeze-dried and a process in which thesolution containing aclacinomycins is freeze-dried after addition of atleast one substance selected from the group consisting ofdeoxy-ribonucleic acid, glycerol, sugars, amino acids, inorganic andorganic acids.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows ultraviolet absorption spectrum of aclacinomycin A inmethanol, 0.1 N HCl-methanol and 0.1 N NaOH-methanol.

FIG. 2 is the infrared absorption spectrum of aclacinomycin A inpotassium bromide.

FIG. 3 is the NMR spectrum of aclacinomycin A in CDCl₃ (100 MHz).

FIG. 4 is ultraviolet absorption spectrum of aclacinomycin B inmethanol, 0.1 N HCl-methanol and 0.1 N NaOH-methanol and

FIG. 5 shows the infrared absorption spectrum of aclacinomycin B inpotassium bromide.

FIG. 6 is the NMR spectrum of aclacinomycin B in CDCl₃ (100 MHz).

DETAILED DESCRIPTION

Aclacinomycin A and B markedly prolong the lives and improve thecondition of mice inoculated with leukemia L1210 and P388 and lymphoma6C3HED cells, and exhibit high lethal dose (LD₅₀) on rats, mice andrabbits and extremely low cardiotoxicity on hamsters. More particularly,the substances caused a remarkable inhibitory effect on the growth ofvarious tumor cells in culture resulting from specific inhibition on RNAsynthesis, and have also strong antimicrobial activity against bacteriaand yeasts.

Aclacinomycin A and B are obtained as yellow microcrystalline powderswhich have the empirical formulae of C₄₂ H₅₄ O₁₅ N and C₄₂ H₅₂ O₁₅ N,respectively, and yield aklavinone, rhodosamine, 2-deoxyfucose,1-deoxypyrromycin and cinerulose A from aclacinomycin A, and aklavinone,rhodosamine, 2-deoxyfucose, 1-deoxypyrromycin and the same methylateddisaccharide as cinerubin B from aclacinomycin B on acid hydrolysis orpartial hydrolysis in methanol, said substances being soluble inmethanol, ethanol, chloroform, acetone, ethyl acetate, benzene and thelike, slightly soluble in water, but insoluble in hexane, cyclohexane,ether and petroleum ether, exhibiting characterisitc absorption maximain the infrared region when pelleted with potassium bromide and inultraviolet and visible light as shown in FIGS. 1, 2 4 and 5 having NMRabsorption spectra substantially as shown in FIGS. 3 and 6, havingspecific rotation in chloroform and melting point respectively of +29°and 129° to 135° C. for aclacinomycin A and +3° and 135° to 145° C. foraclacinomycin B, respectively.

There is further provided according to the present invention the processfor the production of the antitumor antibiotics aclacinomycins A and Bwhich comprises culturing a strain of Streptomyces galilaeus in anaqueous medium containing carbon sources and nitrogen sources underaerobic conditions until a substantial amount of aclacinomycins isaccumulated in said solution, and then recovering said aclacinomycinsfrom said solution using the methods of adsorption to ion-exchanger,adsorbent siliceous earth material and synthetic adsorbents, solventextraction, solvent precipitation, counter-current distribution, etc.and combinations of these methods.

Although a number of anthracycline glycoside antibiotics are well known,e.g. adriamycin, daunomycin, carminomycin, rhodomycin, pyrromycin,aklavin etc., the aclacinomycins produced by the process of thisinvention are clearly different from any of them in the characteristicsof molecular formula, degradation products on acid hydrolysis,ultraviolet, visible, infrared and NMR absorption spectra and the like,as described above. Moreover, their high antitumor activity andremarkably low cardiotoxicity are characteristics not exhibited by anyof the anthracycline glycoside antitumor antibiotics.

The organism producing the antibiotics aclacinomycins A and B of thepresent invention was isolated from a soil sample collected at Osaki,Shinagawa-ku, Tokyo, Japan. A culture of MA144-M1 was deposited in theAmerican Type Culture Collection, Rockville, Maryland and in theFermentation Research Institute, Japan, and added to their permanentcollections of microorganisms as A.T.C.C. No. 31133 and FERM No. 2455,respectively.

The strain No. MA144-M1 has the following properties:

1. Morphological properties:

Under the microscope, open spirals are observed to develop well frombranched substrate mycelia. No whorls, and mature spore chain ismoderately long with more than ten spores. The spores are ellipsoidaland measured 0.4 to 0.8μ × 0.8 to 1.6μ, and its surface is smooth.

2. Properties on various media:

The description in parenthesis follows the color standard "Color HarmonyManual" published by Container Corporation of America, U.S.A.

a. On glucose-aspargine agar, incubated at 27° C. light yellowish browngrowth (3gc, Lt. Tan); no aerial mycelium; no soluble pigment.

b. On sucrose-aspargine agar, incubated at 27° C. colorless or lightyellowish brown growth (3gc, Lt. Tan); no aerial mycelium; no solublepigment.

c. On glycerol-aspargine agar (ISP medium No. 5), incubated at 27° C.:yellowish orange (4ic, SUntan) to brown (51 g., Cocoa Brown) growth;white to light gray (2fe, Covert Gray) aerial mycelium; brown solublepigment.

d. On starch-inorganic salts agar (ISP medium No. 4), incubated at 27°C.: Pale orange (3ea, Lt. Melon Yellow) to pale yellowish brown (3ie,Camel) growth; light gray (2fe, Covert Gray) to gray (e, Gray) aerialmycelium; brown soluble pigment.

e. On tyrosine agar (ISP medium No. 7), incubated at 27° C.: Brownishgray (3li, Beaver) to brown (41g, Toast tan) aerial mycelium, blacksoluble pigment.

f. On nutrient agar, incubated at 27° C.: Colorless to grayish browngrowth; no aerial mycelium, brown soluble pigment.

g. On yeast extract-malt extract agar (ISP medium No. 2), incubated at27° C.: Light brown (41e, Maple) to brown (4ng, Lt. Brown) growth; lightgray (3fe, Silver Gray) to gray (3ih, Beige Gray) aerial mycelium; brownsoluble pigment.

h. On oatmeal agar (ISP medium No. 3), incubated at 27° C.; colorless topale yellowish brown (2gc, Bamboo) growth; light gray (3fe, Silver Gray)aerial mycelium; brown soluble pigment.

i. On glycerol-nitrate agar, incubated at 27° C.: Colorless to paleyellowish brown (3gc, Lt. Tan) or light olive gray (2 db, parchment)growth; no aerial mycelium; no soluble pigment.

j. On starch agar, incubated at 27° C.: Pale yellowish brown (3gc, Lt.Tan) growth; gray (e, Gray) aerial mycelium; slight brown solublepigment.

k. On calcium malate agar, incubated at 27° C.: Colorless growth;grayish white (b, Oyster white) to light brownish gray (3dc, Natural)aerial mycelium; no soluble pigment.

l. On gelatin stab, incubated at 20° C.: Pale brown to pale yellowishbrown growth; white aerial mycelium; brown soluble pigment.

m. On glucose-peptone-gelatin stab, incubated at 27° C; Pale brown tobrown growth; no aerial mycelium; brown soluble pigment.

n. On skimmed milk, incubated at 37° C.: Pale brown to brown growth; noaerial mycelium; brown soluble pigment.

3. Physiological properties:

a. Growth temperature was examined on maltose-yeast extract agar(maltose 1.0%, yeast extract 0.4%, agar 3.5%, pH 6.0) at 20, 24, 27, 30,37 and 50° C. Optimal temperature for the growth is 27° to 37° and nogrowth at 50° C.

b. Gelatin liquefaction on 15 % gelatin stab at 20° C.: and onglucose-peptone-gelatin stab at 27° C.: On the former medium, gelatinliquefaction was observed weakly on the 14 days-incubation, but on thelatter weak or moderate liquefaction after 7 days incubation.

c. Starch hydrolysis on starch-inorganic salts agar at 27° C: Weakhydrolysis was observed after 5 days incubation.

d. Peptonization and coagulation of skimmed milk at 37° C: Moderate tostrong peptonization began after 5 days incubation and finished onaround 17 days. No coagulation.

e. Melanin formation on tyrosine agar (ISP medium No. 7), tyrosine-yeastextract broth (ISP medium No. 1) and peptone-yeast extract-ferrous agar(ISP medium No. 6) at 27° C.: positive on all media.

f. Liquefaction of calcium malate on calcium malate agar at 27° C.:Strongly positive.

g. Nitrate reduction on peptone agar containing 1.0% sodium nitrate (ISPmedium No. 8) at 27° C.: Positive.

h. Utilization of carbohydrates of Pridham-Gottlieb basal medium (ISPmedium No. 9), incubated at 27° C.: Abundant growth with L-arabinose,D-xylose, glucose, D-fructose, sucrose, inositol, L-rhamnose andraffinose; no growth with D-mannitol.

Summarizing the above characteristics of No. MA144-M1, the strainbelongs to the genus Streptomyces and chromogenic type, and producesbrown soluble pigment on various agar media. Aerial mycelium forms openspirals, but no whorls. The spore surface is smooth. The growth onvarious media is found to be pale yellowish brown to brown in general,but olive in a few medium, and aerial mycelium is light gray. Nitrate isreduced to nitrite. Proteolytic action is weak to moderate and starchhydrolysis is relatively weak. Melanin is produced on tyrosine agar,tyrosine-yeast extract broth and peptone-yeast extract-ferrous agar.

Among known species of Streptomyces, strain No. MA-144-M1 resemblesStreptomyces galilaeus. Reference 1: Archiv fur Mikrobiolgie, 31, 356,1958. Reference 2: International Journal of Systematic Bacteriology, 22,298, 1972. With particular attention to differentiation based on themorphology, color of the aerial mycelium and other physiologicalproperties, the difference between the present strain and the standardstrain of S. galilaeus ISP 5481 obtained was investigated by parallelcultures. The results are as follows:

    __________________________________________________________________________                             Streptomyces                                                                  galilaeus                                                                             Reference                                                                             Reference                                            MA144-M1 ISP 5481                                                                               1       2                                   __________________________________________________________________________    Spore surface   smooth   smooth  smooth  smooth                               Spore chain     open spirals                                                                           spirals spirals spirals                              Aerial mycelium light gray                                                                             light gray                                                                            grayish white                                                                         gray                                                                  to gray                                      Growth          pale yellowish                                                                         pale yellowish                                                                        pale yellow                                                                           grayish yellow                                       brown to brown,                                                                        brown-grayish                                                                         to deep ruby                                 yellowish                                                                                     sometimes light                                                                        brown-light                                                                           red     brown-olive brown                                    olive    olive gray                                           Soluble pigment brown    brown   pale brown                                                                            pale yellow                          Melanin formation:                                                             ISP medium No. 1                                                                             positive positive                                                                              positive                                                                              positive                              ISP medium No. 6                                                                             positive probably                                                                              positive                                                                              positive                              ISP medium No. 7                                                                             positive positive                                                                              positive                                                                              positive                             Starch hydrolysis                                                                             weak     weak    weak     *                                   Liquefaction of gelatin:         weak     *                                    Gelatin stab   weak     weak                                                  Glucose-peptone-                                                                             weak to  weak to                                               gelatin stab   moderate moderate                                             Peptonization of milk                                                                         moderate to                                                                            Moderate                                                                              negative                                                                               *                                                   strong                                                        Coagulation of milk                                                                           negative negative                                                                              weak     *                                   Nitrate reduction                                                                             positive positive                                                                               *       *                                   Utilization of carbohydrates:     *                                            L-Arabinose    positive positive        positive                              D-Xylose       positive positive        positive                              Glucose        positive positive        positive                              D-Fructose     positive positive        positive                              Sucrose        positive positive        positive                              Inositol       positive positive        positive                              L-Rhamnose     positive positive        positive                              Raffinose      positive positive        positive                              D-Mannitol     negative negative        negative                             __________________________________________________________________________     * no data                                                                

From the results, the present strain agrees very closely with S.galilaeus ISP 5481 in morphology and color of the growth and mycelium onvarious media and physiological properties. Furthermore, similarity ofboth strains exists in the fermentation products; that is, cinerubinwhich can be produced by S. galilaeus is one of the by-products of thepresent strain. Thus, strain No. MA144-M1 can be identified asStreptomyces galilaeus.

Since the Streptomyces are easily mutatable naturally or artificially,S. galilaeus No. MA144-M1 in the present invention includes the typicalstrain described above and all natural and artificial variants andmutants thereof. That is, S. galilaeus No. MA144-M1 of the presentinvention includes all strains producing aclacinomycin. As with theknown antibiotics it is anticipated that higher production ofaclacinomycin can be achieved by the selection of highly productivestrains after single colony selection, by the treatment of anaclacinomycin-producing strain with various mutagens, or by the geneticprocedures of recombination, transformation or transduction.

Aclacinomycins are produced by the cultivation of S. galilaeus undersuitable conditions. The general procedures used for the cultivation ofother actinomycetes are applicable to the cultivation of S. galilaeus. Afermentation broth containing aclacinomycin is prepared by inoculatingspores or mycelia of the aclacinomycin-producing organism into asuitable medium and then cultivating them under aerobic conditions.Although cultivation on a solid medium is possible for the production ofaclacinomycin, submerged aerobic culture is especially advantageous forproduction of large quantities of the antibiotics. Media consisting ofknown kinds of nutritional sources for actinomycetes are useful. Themedium preferably contains commercially available products such asglycerol, glucose, starch, dextrin, maltose, molasses, oils, fats,lipids and the like as carbon sources in either purified or crude state,and as the nitrogen sources, commercially available products such assoybean meal, malt extract, peptone, yeast extract, distiller'ssolubles, fish meal, gluten meal, corn steep liquor, cottonseed flour,casein, hydrolyzed protein substances, nitrates, ammonium salts, ureaand the like, and inorganic salts such as sodium chloride, potassiumchloride, potassium phosphate, magnesium sulfate, calcium carbonate andtrace amounts of heavy metal salts such as copper, zinc, manganese,iron, and the like. In the aerated submerged culture an antifoam such asliquid paraffin, soybean oil, fat or silicone is used. Any fermentationtemperature can be employed within the range, 20° to 35° C., in which anaclacinomycin-producing organism can grow, although the most preferredrange of temperature is 25° to 30° C. The pH of the culture mediumranges from 5 to 8.0.

Unless it is otherwise specified, the method of cultivation and theassay method are as follows:

1. Shaking culture: 100 ml. of medium in a Sakaguchi flask or 50 ml. ofmedium in an Erlenmeyer flask of 500 ml. is sterilized at 120° C. for 15minutes. Spores or mycelia of an aclacinomycin-producing organism areinoculated into the sterilized medium from an agar slant culture byplatinum loop and cultivated at 28° C. for 4 days on a reciprocal orrotary shaker.

2. Tank culture: 100 liters of medium are prepared in a 200 literfermenter and sterilized at 120° C. for 15 minutes. The sterilizedmedium is inoculated with 2 liters of cultured broth which waspreviously shake-cultured for 2 days. The fermentation in the tankproceeds under aeration of 50 liters sterile air per minute, withstirring at 160 r.p.m. for 2 days. Silicone oil and liquid paraffin areused for defoamer.

3. Assay of aclacinomycin-TLC-chromatoscanner method; Aclacinomycins Aand B extracted from the cultured broth, mycelia or culture filtrate byacetone, ethyl acetate or chloroform-methanol mixture are spotted on thesilica gel plate (Kieselgel 60F₂₅₄, Merck Co.) with 5 mm in diameter.After 15 minutes saturation in the developing vessel, TLC plate isdeveloped in acetone ascending to 15 cm at room temperature. Spotscorresponding to aclacinomycin A and B are determined by optical densityat 430 nm using a Shimadzu Dual-Wave length TLC Scanner, Model CS-900,under a certain condition, Zig-Zag scanning at 430 nm-700nm, 1.25 × 1.25nm slit, 10 mm/min scan and chart speed. The standard curves of purifiedaclacinomycin A and B are previously prepared ranging from 0.2 to 15mcg./spot.

The aclacinomycin-producing strain was first shake-cultured in thefollowing medium.

The basal medium consisted of 1% glucose, 1% potato starch, 1.5%"Prorich" (soybean meal, the product of Ajinomoto Co.), 0.1% KH₂ PO₄,0.1% MgSO₄.7H₂ O, 0.3% NaCl, 0.0007% CuSO₄.5H₂ O, 0.0001% FeSO₄.7H₂ O,0.0008% MnCl₂.4H₂ O and 0.0002% ZnSO₄.5H₂ O, and adjusted to pH 7.0.

The cultured broth at the 4th day exhibited the accumulation of 45mcg./ml. of aclacinomycin A and 15 mcg./ml. of aclacinomycin B.

Aclacinomycin A and B were produced in media containing various carbonand nitrogen sources under shaking conditions as shown by examplesdescribed as follows:

1. Various carbon sources were added to the basal medium consisting of1.5% "Prorich" and other salts as follows:

    __________________________________________________________________________                     Aclacinomycin A present at indicated time                                     2 days  3 days  4 days                                                        pH  mcg/ml                                                                            pH  mcg/ml                                                                            pH  mcg/ml                                   __________________________________________________________________________    2% glucose       7.3 27  7.4 38  8.0 41                                       2% maltose       6.6 15  6.0 28  7.0 32                                       2% sucrose       7.3 32  7.7 39  8.2 40                                       2% soluble starch                                                                              7.4 35  7.4 28  7.9 33                                       2% potato starch 7.2 36  7.6 33  8.1 42                                       1% glucose + 1% potato starch                                                                  6.3 32  7.7 34  8.3 43                                       1% glucose + 1% maltose                                                                        7.4 26  6.7 28  7.7 30                                       1% glucose + 1% sucrose                                                                        7.3 26  7.6 30  7.8 32                                       1% glucose + 1% soluble starch                                                                 7.1 37  7.9 37  8.4 39                                       1% glucose + 1% glycerol                                                                       4.9 36  6.3 36  8.1 41                                       __________________________________________________________________________

2. Various nitrogen sources were added to the basal medium consisting of1% glucose, 1% potato starch and other salts as follows:

    __________________________________________________________________________                            Aclacinomycin A present at indicated time                                     2 days   3 days   4 days                                                      pH  mcg/ml.                                                                            pH  mcg./ml.                                                                           pH  mcg./ml.                        __________________________________________________________________________    1.5% "Prorich" (soybean meal, Ajinomoto Co.)                                                          6.3 34   7.0 48   8.0 45                              1.5% "Meat" (soybean meal, Ajinomoto Co.)                                                             5.8 37   7.1 54   8.1 48                              1.5% malt extract       6.2  9   6.6  7   6.6 13                              1.5% yeast extract      5.4 45   7.7 43   8.5 40                              1.5% casamino acid      6.9 18   8.0 21   8.3 22                              1.5% beef extract       5.6  9   7.2 32   8.2 36                              1.5% polypeptone        6.2 38   8.2 28   8.6 23                              __________________________________________________________________________

3. When the concentrations of carbon and nitrogen sources in the basalmedium were varied, aclacinomycin A and B were accumulated as follows:

    ______________________________________                                                       Carbon source %                                                Glucose          1      2         3                                           Potato starch    1      2         3                                           ______________________________________                                                    * A  B      A      B    A    B                                    ______________________________________                                        "Prorich"                                                                              1.5%     33     23   36   19   --   --                                        2.5%     31     39   39   28   51   34                               "Meat"   1.5%     32     19   48   31   --   --                                        2.5%     17     24   43   28   58   29                               ______________________________________                                          *aclacinomycin A or B: mcg./ml.                                         

The above results are merely examples, and it can be said that carbonsources such as glucose, starch, maltose and sucrose are favorable forthe production of aclacinomycin and nitrogen sources such as soybeanmeal, yeast extract, beef extract and peptone are suitable.

When the fermentation was carried out at 28° C. with shaking using oneof the suitable media at pH 6.9, i.e. 2% glucose, 2% potato starch, 2.5%"Meat", 0.1% KH₂ PO₄, 0.1% MgSO₄.7H₂ O, 0.0007% CuSO₄.5H₂ O, 0.0001%FeSO₄.7H₂ O, 0.0008% MnSO₄.4H₂ O and 0.0002% ZnSO₄.5H₂ O, the pH of themedium dropped to 5.25 to 5.10 in 20 hours and the growth of myceliumincreased at 10 hours after inoculation. Thereafter, pH rose to 7.0 to7.5 on 3 days and aclacinomycin A and B reached a maximum, i.e. 46mcg./ml. of A and 23 mcg./ml. of B in the broth. In order to isolateaclacinomycin, the antibiotic may be extracted with a suitable solventeither from the cultured broth "in toto" without filtering the myceliummass or from the mycelium and the culture liquid previously separated byfiltration. Most of the antibiotics exist in the filtration cake whichconsists of the mycelium admixed with the diatomaceous earth.Thereafter, the filtration cake is pulped and stirred in an organicsolvent for the extraction of antibiotics. Suitable solvents arealcohols such as methanol, ethanol, butanol, ketones such as acetone,methyl ethyl ketone; halogenated hydrocarbons such as chloroform,methylene chloride, benzene or aqueous solutions of organic or inorganicacids such as acetic acid, hydrochloric acid, sulfuric acid. Preferredare acetone, ethyl acetate, mixtures of organic solvents such asalcohols and water-immiscible ketones and aqueous solution of inorganicor organic acids.

In order to extract the antibiotics in the filtrate, it is advantageousto add to the weakly acidified or neutralized filtrate a double volumeof a water-immiscible organic solvent such as butanol, chloroform, ethylacetate, butyl acetate or benzene. Among the separation methods foraclacinomycin, the effective one may be carried out by counter currentdistribution chromatography using silicic acid, alumina, Sephadex LH20(Pharmacia Fine Chemicals AB, Uppsala, Sweden) or synthetic adsorbents,adsorption to active carbon and the like, and the combination of solventextraction and these methods.

The antibiotics can be directly extracted from cultured broth by theabove-mentioned methods without the separation of mycelium.

After concentrating in vacuo, the extracts are made at a pH between 5 to7 and re-extracted with a water-immiscible solvent, for example,n-butanol, amyl alcohol, ethyl acetate, chloroform, benzene, methylenechloride, methyl propyl ketone and the like. From the water-immiscibleorganic solution the active crude substance is obtained as a yellowpowder by concentration in vacuo to a small volume and precipitationwith low polar solvents, for example, saturated hydrocarbons such asn-hexane, cyclohexane and petroleum ether or ethers such as diethylether and dipropyl ether.

To remove other pigmented substances such as cinerubin A and B from theactive crude substance and to obtain pure aclacinomycin A and B, furtherpurification may be carried out by column chromatography using variousadsorbents such as silicic acid, alumina, Sephadex LH20, byion-exchangers such as weakly acidic resins andpolystyrene-divinylbenzene cross-linked matrix resins such as AmberliteXAD, by chelation with various metals such as cupric, ferrous, ferric,calcium and magnesium ions, and by combinations of at least one or moreprocesses selected from the methods of chelation with metal ions,solvent precipitation, solvent extraction, counter current distributionetc.

For example, when crude aclacinomycins A and B was dissolved in a smallamount of chloroform, subjected to a silicic acid column and then elutedwith chloroform-methanol mixture, aclacinomycin B fraction was elutedfirst with a 50:1 chloroform-methanol mixture and then aclacinomycin Awas eluted with above mixture of 30:1 to 20:1. The eluates ofaclacinomycins A and B were separately concentrated in vacuo, and werefurther separated completely from cinerubin A and B and a small amountof impure pigmented substances by means of synthetic adsorbent columnchromatography, i.e., Column Lite (Al₂ O₃.MgO.2SiO₂.4H₂ O, Fuji ChemicalCo.), Floridil (Activated magnesium silicate, Floridin Co.),hydroxylapatite, or of the addition of 1 × 10.sup.⁻³ to 1 × 10.sup.⁻² MCuSO₄, FeCl₃, FeSO₄ or MgSO₄ to make chelate complex of cinerubin A andB, followed by Sephadex LH 20 column chromatography. The resultingsolutions of pure aclacinomycin A and B can be concentrated in vacuo todryness, and also be lyophilized alone or with at least one substanceselected from serum albumin, globulin, gelatin, glycerol, sugars, aminoacids, inorganic or organic acids etc.

Aclacinomycins A and B obtained by a combination of the above-mentionedmethods and described in the examples below was demonstrated to be pureand uniform by single spot on the thin-layer chromatography usingvarious solvent systems, constant melting point and dextrorotation,elementary analysis, characteristic peaks on ultraviolet, visible, NMRand infrared absorption spectra, and has the following properties:

Aclacinomycin A:

Weakly basic and lipophilic yellow powder or microcrystalline powder.Elementary analysis yields the following values:

    ______________________________________                                        Found    C = 62.37%      C = 62.05%                                                    H =  6.67%      H =  6.69%                                                    0 = 29.38%      O = 29.52%                                                    N =  1.82%      N =  1.72%                                           Molecular weight = 813.                                                       ______________________________________                                    

The melting point and dextrorotation ( [α]_(D) ²⁴) of its 1% solution inchloroform were 129° to 135° C. and +29° respectively. Its absorptionspectra in the ultraviolet and in the visible ranges (FIG. 1) show bandsat the following wave-lengths:

in methanol:

at 229.5 nm, E₁ cm^(1%) = 540

258 nm, E₁ cm^(1%) = 301

290 nm, E₁ cm^(1%) = 128

434 nm, E₁ cm^(1%) = 147

in 0.1 N HCl-methanol:

at 229.5 nm, E₁ cm^(1%) = 578

258 nm E₁ cm^(1%) = 329

290 nm E₁ cm^(1%) = 141

434 nm E₁ cm^(1%) = 164

in 0.1 N NaOH-methanol:

at 239.5 nm, E₁ cm^(1%) = 678

288 nm, E₁ cm^(1%) = 232

314 nm, E₁ cm^(1%) = 156

523 nm, E₁ cm^(1%) = 170

When pelleted with potassium bromide, characteristic absorption bands inthe infrared spectrum (FIG. 2) are exhibited at the following wavenumbers is cm.sup.⁻¹ : 3300, 3080, 2940, 2860, 2760, 1740, 1640, 1620,1540, 1460, 1450, 1415, 1385, 1295, 1250, 1220, 1195, 1165, 1125, 1105,1090, 1010, 960, 930, 890, 840, 815, 760, 730, 580 and 450. Proton-NMRspectrum was recorded on a Varian XL-100-15 spectrophotometer operatingin the Fourier transform mode at 100 MHz as shown in FIG. 3.

The antibiotic aclacinomycin A is soluble in methanol, ethanol,chloroform, ethyl acetate, acetone, benzene, dimethyl sulfoxide (DMSO)and methyl cellosolve, is slightly soluble in water, but is insoluble inethyl ether, hexane, cyclohexane and petroleum ether. The aqueoussolution is yellow and turns to reddish brown in concentrated sulfuricacid. With alcoholic magnesium acetate this solution shows a purplishred coloring and turns to red purple on alkalinization.

Aclacinomycin B:

Weakly basic, lipophilic and yellow powder or microcrystalline powder.Elementary analysis yields the following values:

    ______________________________________                                                           Calcd. for C.sub.42 H.sub.52 NO.sub.15                     ______________________________________                                        Found    C = 62.21%      C = 61.87%                                                    H =  6.46%      H =  6.29%                                                    O = 29.60%      O = 29.80%                                                    N =  1.73%      N =  1.89%                                           Molecular weight = 811                                                        ______________________________________                                    

The melting point is 135° to 145° C. and the dextrorotation ([ α]_(D) ²⁴in 1% chloroform) is +3°. The absorption spectra in the ultraviolet andin the visible ranges (FIG. 4) show bands at the following wave-lengths:

in methanol:

at 229.5 nm, E₁ cm^(1%) = 452

257.5 nm, E₁ cm^(1%) = 261

290 nm, E₁ cm^(1%) = 120

433 nm, E₁ cm^(1%) = 120

in 0.1 N HCl-methanol:

at 229.5 nm, E₁ cm^(1%) = 465

257.5 nm, E₁ cm^(1%) = 261

290 nm, E₁ cm^(1%) = 125

433 nm, E₁ cm^(1%) = 134

in 0.1 N NaOH-methanol:

at 238 nm, E₁ cm^(1%) = 440

286-289 nm, E₁ cm^(1%) = 142

315 nm, E₁ cm^(1%) = 97

525 nm, E₁ cm^(1%) = 135

Characteristic absorption bands in the infrared spectrum (FIG. 5) areexhibited at the following wave numbers in cm.sup.⁻¹ (KBr): 3300, 3070,2940, 2850, 2750, 1740, 1640, 1620, 1540, 1465, 1445, 1410, 1380, 1290,1245, 1215, 1160, 1120, 1095, 1050, 1010, 960, 920, 880, 840, 820, 750,725, 700, 600, 580 and 450. NMR spectrum of aclacinomycin B is shown inFIG. 6.

The antibiotic is soluble in methanol, ethanol, chloroform, ethylacetate, acetone and acidic water, is slightly soluble in water, but isinsoluble in hexane, cyclohexane, and ethers. The aqueous solution isyellow and turns to deep reddish purple in concentrated sulfuric acid.This solution shows purplish red in alcoholic magnesium acetate, andgives red purple on alkalinization.

Aclacinomycins A and B have the following Rf values on silica gelthin-layer chromatograms using various solvent systems.

    ______________________________________                                                        Rf values of aclacinomycin                                    ______________________________________                                                        A         B                                                   ______________________________________                                        Chloroform:methanol                                                             20:1            0.36        0.71                                               5:1            0.88        0.90                                            Acetone           0.43        0.72                                            Acetone:hexane                                                                   1:1            0.15        0.49                                            Benzene:methanol                                                                 1:1            0.86        0.93                                            ______________________________________                                    

On partial hydrolysis or methanolysis with dilute hydrochloric acid orin methanol containing 5% hydrochloric acid for 1 to 2 hours at roomtemperature, aclacinomycins A and B give methylated disaccharides and1-deoxypyrromycin. Physicochemical properties such as absorption spectraof infrared, ultraviolet, visible ranges and NMR, melting point anddextrorotation of the methylated disaccharide obtained fromaclacinomycin B coincided fully with those of the methylateddisaccharide obtained from cinerubin B by partial acid hydrolysis(Helvetica Chimica Acta, 55, 467-480, 1972), and 1-deoxypyrromycin wasalso identified with the data of rhodomycin (Chem. Ber. 38, 1762, 1955,Naturwiss., 48, 716, 1961), cinerubin (Chem. Ber. 92, 1868, 1959) andpyrromycin (Chem. Ber. 92, 1904, 1959) on the basis of NMR spectrum,mass spectrum of aglycone, and the presence of rhodosamine in the acidhydrolysate.

Aclacinomycins A and B decompose into a neutral aglycone and two orthree reducing sugars on hydrolysis with 0.3 N sulfuric acid for 3 hoursat 85° C. The aglycone forms orange yellow crystals melting 171° to 174°C. and containing oxygen, carbon and hydrogen. Elementary analysis,infrared, ultraviolet and NMR spectra, fragment ion peaks of massspectrum and other properties demonstrated that the aglycone ofaclacinomycin A and B is aklavinone (Tetrahedron Letters, 8, 28, 1960,Naturwiss., 47, 135, 1960, Naturwiss. 42, 154, 1955, Naturwiss. 50, 92,1963). Furthermore, according to the comparative analysis of sugars inthe acid hydrolysates of aclacinomycin A and B and cinerubins A and B bythin-layer chromatography, aclacinomycin A has the same rhodosamine,2-deoxyfucose, and cinerulose A as cinerubin A and aclacinomycin B hasthe same rhodosamine and 2-deoxyfucose as cinerubin B.

From the results mentioned above, the structures of aclacinomycins A andB in the present invention are as follows: ##SPC1##

Aclacinomycins A and B (according to their physiochemical properties andstructures as described above and by comparison with known antibioticsreported in the publications) belong to the group of anthracyclineglycosides and are similar to cinerubins A and B, rutilantine, aklavin,requinomycin and galirubins.

Cinerubin and rutilantine are different from the present antibiotics inits aglycone, ε-pyrromycinonone (Tetrahedron Letters, 16, 17, 1959).Among the antibiotics having aklavinone aglycone, requinomycin (J.Antibiotics, 25, 393, 1972) and aklavin (J. Bacteriol., 72, 90, 1956)are not identical with the present antibiotics in the sugar moiety andmolecular formula based on elementary analysis. Galirubins which areproduced by Streptomyces galilaeus are most similar to the presentantibiotics. K. Eckardt has reported that four components of galirubin(galirubin D, C, B and A) have been isolated from the mycelium of S.galilaeus and that galirubin A was ε-pyrromycinone glycoside, B wasaklavinone glycoside, galirubinone C was ζ-pyrromycinone andgalirubinone D was 7-deoxy aklavinone. In respect of the physicochemicalproperties of galirubin B which has aklavinone as its aglycone, thereport has not clearly presented its detailed purification procedure,chromatographic behavior, melting point, elementary analysis, absorptionspectra of infrared, ultraviolet, visible ranges, and NMR, and onlyelucidated that two sugar spots were detected in the acid hydrolysate ofgalirubin B on the paper chromatogram.

Thus aclacinomycins A and B, having aklavinone aglycone and threesugars, is clearly differentiated from galirubin B as in the abovestated properties. It is verified that the present antibiotics are newsubstances discovered by the present inventors.

Therapeutically useful non-toxic salts and deoxy-ribonucleic acid(DNA)-complexes of the antibiotics aclacinomycins A and B can be derivedfrom organic and inorganic acid, for example, hydrochloric acid,phosphoric acid, sulfuric acid, acetic acid, propionic acid, oleic acid,palmitic acid, citric acid, succinic acid, glutamic acid, pantothenicacid and DNA obtained from calf thymus, HeLa cell, human embryonic cell,E. coli and other animals and microorganisms.

Biological activities of aclacinomycins A and B are as follows:

1. The antimicrobial spectrum of aclacinomycins A and B was determinedby the broth dilution method as follows:

    ______________________________________                                        Antimicrobial spectrum of aclacinomycins A and B                              ______________________________________                                                        Minimum Inhibitory                                                            Concentration (mcg./ml)                                       ______________________________________                                        Test organism     A           B                                               ______________________________________                                        Bacillus subtilis ATCC 6633                                                                     <0.2        <0.2                                            B. cereus ATCC 9634                                                                             <0.2        <0.2                                            B. megaterium     <0.63       <0.2                                            Staph. aureus FDA 209P                                                                          <0.63       <0.2                                            Staph. aureus Smith                                                                             < 0.2       <0.2                                            Sar. lutea ATCC 9341                                                                            <0.2        <0.2                                            Mic. flavus       <0.2        <0.2                                            Cory. bovis 1810  <0.2        <0.2                                            Mycobact. smegmatis ATCC 607                                                                    2.5         5                                               Strept. faecalis  2.5         2.5                                             St. pyogenes NY 5 1.25        1.25                                            Diplo. pneumoniae Type 1                                                                        0.63        0.63                                            Diplo. pneumoniae Type 3                                                                        0.63        0.63                                            E. coli K12       >100        >100                                            Kl. pneumoniae ATCC 10031                                                                       >100        >100                                            Ps. aeruginosa A20229                                                                           >100        >100                                            Can. albicans IAM 4905                                                                          10          10                                              Can tropicalis IAM 4924                                                                         20          20                                              ______________________________________                                    

2. Antitumor effects: Furthermore, the antibiotics aclacinomycin A and Bshow marked inhibitory effects on experimental animal tumors of asciticand solid tumors. This antitumor action can be most significantlydemonstrated in leukemia in mice and hepatomas in rats. For example,when BDF₁ mice weighing 18 to 22 grams are inoculated with 1 × 10⁶ cellsof P388 cells or 5 × 10⁵ cells of L1210 cells intraperitoneally andaclacinomycin A and B and aclacinomycin A-DNA complex are administeredintraperitoneally once daily for 10 days consecutively 24 hours afterthe inoculation, the survival time of the mice is prolonged remarkablyat the rate of over 150% in a dosage range of 0.5 to 5 mg./kg. of bodyweight per day compared with control mice receiving no antibiotic, asshown in the following table:

    ______________________________________                                                              Mean survival                                                      Dose       time       T/C  30 day                                  Antibiotic (mg./kg./day)                                                                            (days)     (%)  survivors                               ______________________________________                                        Aclacinomycin A                                                                          4.0        21.5       215  1/4                                                2.0        30.0       300  4/4                                                1.0        28.0       280  3/4                                                0.5        23.0       230  2/4                                     Aclacinomycin B                                                                          4.0         8.5        85  0/4                                                2.0        17.5       175  0/4                                                1.0        19.0       190  1/4                                                0.5        14.7       147  0/4                                     Aclacinomycin A-                                                                         4.0        19.0       190  1/4                                     DNA complex                                                                              2.0        30.0       300  4/4                                                1.0        30.0       300  4/4                                                0.5        27.3       273  3/4                                     control    --         10.0       --   0/8                                     ______________________________________                                    

When CH3/He mice weighing 20 to 23 grams are inoculated with 1 × 10⁷cells of lymphoma 6C3HED/OG intraperitoneally or with 5 × 10⁶ cells oflymphoma 6C3HED/OG subcutaneously and aclacinomycin A is administeredintraperitoneally once daily for 10 days consecutively 3 hours after theinoculation, aclacinomycin A shows marked inhibition of the growth ofascitic or solid 6C3HED/OG tumor as shown in the following table.

    __________________________________________________________________________                       Aclacinomycin A, Dose: mg./kg./day                         __________________________________________________________________________                       4      2      1      0.5    control                        __________________________________________________________________________    Solid type:                                                                           Tumor weight (mg)                                                                        5023   4633   2501   3695   7045                                   Tumor weight                                                                  Body weight (%)                                                                          19.5   15.1   9.5    12.4   29.6                                   Inhibition (%)                                                                           28.7   34.2   64.5   47.6   --                                     21 day survivors                                                                         0/3    3/3    3/3    3/3    7/9                            Ascitic type:                                                                         MST (days)*                                                                              20.1   31.8   37.5   15.0   20.5                                   T/C (%)**  98     155    183    73     --                                     62 day survivors                                                                         0/3    0/3    1/3    0/3    0/3                            __________________________________________________________________________      *Mean survival time                                                          **Survival ratio: Tested/control                                         

Mice are inoculated intraperitoneally with a suspension of L1210leukemia cells and treated with solutions of different concentrations ofaclacinomycin A and 1,5 and 9 days following the tumor implantation. Thefollowing table, where the obtained results are summarized, shows thataclacinomycin A, administered in doses of 6 and 3 mg./kg./day, hasincreased considerably the mean survival time of the treated mice.

    ______________________________________                                        Dose       Mean Survival Time                                                                             30 day                                            (mg/kg/day)                                                                              (days)           survivors                                         ______________________________________                                        12         16.1             0/5                                               6          21.7             2/5                                               3          23.3             2/5                                               1.5        12.6             0/5                                               Control    10.4              0/10                                             ______________________________________                                    

Donryu rats, grafted with ascitic hepatoma AH44 were treatedintraperitoneally for 10 consecutive days with 2 mg./kg./day ofaclacinomycin A. While the control rats had an average survival time of14.5 days after the tumor implantation, all the treated rats were alive30 days after the experiment.

3. Acute toxicity: LD₅₀ 's upon a single injection of aclacinomycin A orB are summarized in the following table:

    ______________________________________                                        Animal                 LD.sub.50  (mg./kg.)                                   Species  Route         A          B                                           ______________________________________                                        Mouse    intravenous   33.7       16.4                                        Mouse    intraperitoneal                                                                             22.6       13.7                                        Rat      intravenous   32.5       15.0                                        Rat      intraperitoneal                                                                             25-50      10-15                                       ______________________________________                                    

Acute cardiac alterations in the electrocardiograms of hamsters were notinduced by a single intravenous administration of 25 mg./kg. ofaclacinomycin A or B.

4. The antibiotics inhibit the growth of cultured mammalian tumor cells,vaccinia virus in HeLa cells, and specifically the RNA synthesis at anextremely low concentration. When L1210 cells were inoculated in themedium, Rosewell Memorial Park Institute 1640, containing 10% calf serumand there were added various concentrations of aclacinomycin A and B,and as a precursor, ¹⁴ C-thymidine, -leucine or -uridine and thenincubated at 37° C. for 60 min. in the ¹⁴ C-incorporation experiment and3 days in the growth experiment, nucleic acid biosynthesis and cellgrowth were inhibited over 50% below 1 mcg./ml. aclacinomycin in themedium, as shown in the following table:

    __________________________________________________________________________                ID.sub.50 (mcg./ml.)                                                                    Aclacinomycin A-                                                    Aclacinomycin A                                                                         DNA complex*                                                                            Aclacinomycin B                               L1210 cells:                                                                   Growth     0.12      0.08      0.24                                           DNA synthesis                                                                            1.1       --        4                                              RNA synthesis                                                                            0.1       0.2       0.2                                            Protein synthesis                                                                        6.3       --        12                                            Vaccinia virus:                                                                           <10       --        <10                                           __________________________________________________________________________     *Calf thymus DNA, Type V (Sigma Co.) was used.                           

In view of the aforesaid properties of the aclacinomycin A andaclacinomycin B substances, it has been confirmed that these substancesare new antibiotics which are differentiated from any of the knownantibiotics. According to a third aspect of this invention, there isprovided a method for therapeutically treating a living animal, affectedby leukemia, which comprises administering the aclacinomycin A substanceand/or the aclacinomycin B substance to said animal in a dosagesufficient to reduce the affection by leukemia. According to a fourthaspect of this invention, there is further provided a pharmaceuticalcomposition comprising the aclacinomycin A substance and/or theaclacinomycin B substance in an amount sufficient to reduce theaffection by leukemia in vivo, the aclacinomycin A substance and/or theaclacinomycin B substance being in combination with a pharmaceuticallyacceptable carrier. It will be appreciated that the actual preferredamounts of the aclacinomycin substance used will vary according to theparticular compound being used, the particular composition formulated,the mode of application and the particular situs and organism beingtreated. Many factors that modify the action of the drug will be takeninto account by the skilled in the art, for example, age, body weight,sex, diet, time of administration, route of administration, rate ofexcretion, drug combinations, reaction sensitivities and severity of thedisease. Optimal application rates for a given set of conditions can beascertained by the skilled in the art using conventional dosagedetermination tests in view of the above guidelines.

Actual examples for the production and purification of aclacinomycins Aand B are described below. The following examples are merelyillustrative and it should be understood that our invention is notlimited to these examples.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Example 1

An aqueous medium having the following composition was prepared:

    ______________________________________                                                        Percent                                                       ______________________________________                                        Potato starch     1                                                           glucose           1                                                           "Prorich"         1.5                                                         KH.sub.2 PO.sub.4 0.1                                                         K.sub.2 HPO.sub.4 0.1                                                         MgSO.sub.4.7H.sub.2 O                                                                           0.1                                                         NaCl              0.3                                                         Minerals*         0.125                                                       silicone (KM75)   0.05                                                        pH                7.0                                                         ______________________________________                                         *Minerals: CuSO.sub.4.5H.sub.2 O 2.8 g, FeSO.sub.4.7H.sub.2 O 0.4 g,          MnCl.sub.2.4H.sub.2 O 3.2 g, ZnSO.sub.4.7H.sub.2 O 0.8 g, in 500 ml.          water.                                                                   

100 ml. of this medium was sterilized at 120° C. for 15 min. in a 500ml. Sakaguchi-shaking flask which was inoculated from an agar slantculture of Streptomyces galilaeus MA144-M1 by platinum loop. Incubationproceeded for 48 hr. at 28° C. on a reciprocal shaker. Ten liters of thepreviously sterilized medium in a 20 liter stainless steel har fermenterwere aseptically inoculated with 200 ml. of the above seed cultures.Fermentation was carried out at 28° C. for 32 hours with agitation (240rpm) and aeration (51/min.). The cultured broth obtained was adjusted topH 4.5, mixed with an adsorbent siliceous earth material and filteredfrom the mycelium. The filtrate and cake obtained thereby were extractedseparately. The cake was suspended in acetone (3 liters per kg. wetcake), stirred for 2 hrs. and filtered, and the cake was furtherextracted with acetone once again. The extracts thus obtained wereevaporated to one tenth volume in vacuo. The culture filtrate wasadjusted to pH 6.8 and extracted twice with one third volume of ethylacetate, and the ethyl acetate extracts were concentrated to one tenthvolume in vacuo.

Twenty grams of the resulting oily substances were mixed with 20 gramsof silicic acid (Mallinckrodt Chem. Co.), applied to a column 40 cm. inlength and 4.5 cm. in diameter filled with silicic acid, and eluted witha benzene-acetone-methanol mixture. The initial eluate which eluted witha 1:1:0 mixture was discarded and the active fractions eluted with 1:3:0and 1:3:0.3 mixtures were collected and concentrated to dryness invacuo. 11.5 g. of this crude substance was then dissolved in a smallamount of ethyl acetate and applied to the same silicic acid column asabove. After discarding the initial eluates by the 1:1 and 2:1benzene-acetone mixtures, aclacinomycin B fractions were first elutedwith the above mixtures of 1:3 and 1:5 ratio, and aclacinomycin Afractions were then eluted with the 1:5:0.5 and 1:5:1benzene-acetone-methanol mixtures. The eluates were dried over anhydroussodium sulphate and concentrated to dryness in vacuo. 4.8 g. of crudeaclacinomycin A and 3.5 g. of aclacinomycin B were obtained as yellowpowder.

Example 2

2.0 g. of crude aclacinomycin B obtained as in Example 1 were mixed with3 g. of silicic acid, subjected to a column 15 cm in length and 2 cm indiameter filled with 25 g. of silicic acid, and the aclacinomycin Bfraction was eluted with chloroform containing 1% methanol andconcentrated to a small volume in vacuo. From the concentrated solutionby adding some n-hexane, 40 mg. of aclacinomycin B was obtained asyellow powder.

Example 3

2.0 g. of crude aclacinomycin A obtained as in Example 1 were dissolvedin a small amount of chloroform, applied to a column 20 cm in length and20 cm in diameter filled with 30 g. of silicic acid. After eluting offthe pigments containing aglycone and aclacinomycin B and otherimpurities with chloroform and 1.5% methanol-containing chloroform,aclacinomycin A fractions were eluted with 2% methanol-containingchloroform, and concentrated to dryness in vacuo. 53 mg. of yellowpowder of aclacinomycin A was obtained.

Example 4

Crude aclacinomycin A or B obtained as in Example 1 and 3 still containsa small amount of cinerubin A or B which was produced in the brothcultivated as in Example 1, and the impure cinerubin A or B can beremoved by chelation with various metal ions.

250 mg. of crude powder of aclacinomycin A obtained as in Example 4 wasdissolved in 20 ml. chloroform, and there was added 20 ml. of 1% CuSO₄solution. After the solution was shaken strongly and stood for 1 hr, tothe chloroform phase separated from the aqueous phase there was added10.sup.⁻³ M EDTA (ethylenediamine tetraacetic acid) solution and thatmixture was shaken vigorously for 1 min. and then the chloroform phasewas separated and washed with water twice by shaking. The chloroformextract was dried over anhydrous sodium sulphate and concentrated invacuo, and the addition of some n-hexane caused the precipitation of 120mg. of pure aclacinomycin A.

Example 5

300 mg. of crude aclacinomycin B obtained as in Example 1 was dissolvedin 10 ml. of methyl cellosolve and there was added 1 ml. of 100 mg./ml.CuSO₄ solution. After standing overnight, some precipitates werecentrifuged off, and then the supernatant was concentrated to one-halfvolume in vacuo and chromatographed by using a column 100 cm in lengthand 4 cm in diameter filled with Sephadex LH20 in methanol. The initialimpurities and purplish cinerubin B-Cu complex were discarded andsubsequent yellow fractions were collected, added to 10.sup.⁻³ M EDTAsolution and concentrated to one third in vacuo. Aclacinomycin B wasextracted from the concentrate with chloroform and concentrated todryness in vacuo. 128 mg. of pure aclacinomycin B was obtained.

Example 6

Aqueous medium having the following composition was prepared:

    ______________________________________                                                        Percent                                                       ______________________________________                                        potato starch     2                                                           glucose           2                                                           "Meat"            2.5                                                         KH.sub.2 PO.sub.4 0.1                                                         K.sub.2 HPO.sub.4 0.1                                                         MgSO.sub.4.7H.sub.2 O                                                                           0.1                                                         NaCl              0.3                                                         MnCl.sub.2.4H.sub.2 O                                                                           0.0005                                                      FeSO.sub.4.7H.sub.2 O                                                                           0.0005                                                      silicone          0.05                                                        pH                7.2                                                         ______________________________________                                    

Fifty ml. of this medium was sterilized at 120° C. for 15 min. in a 500ml. Erlenmeyer flask which was inoculated with 1 ml. of frozen culturedbroth of Streptomyces galilaeus MA144-M1. Incubation was carried out 48hours at 30° C. on a rotary shaker. Ten liters of the same medium in a20 liter stainless steel jar fermenter were aseptically inoculated with200 ml. of the above cultures. After incubation at 30° C. for 18 hourswith agitation (300 rpm) and aeration (5 l/min.), ten liters of thecultured broth were added to 600 liters of the above sterilized mediumin a 1 kiloliter stainless steel tank and incubated at 30° C. for 48hours with aeration (200 l/min.) and agitation (180 rpm). The culturedbroth (570 l) obtained was adjusted to pH 5.0 with addition of 250 ml.of 30% H₂ SO₄ mixed with an adsorbent siliceous earth material and the53.5 kg. of cake so obtained was suspended in 70 l of acetone, stirredfor 3 hours and filtered. The cake was further extracted with 85 litersof acetone once again. The extracts were evaporated to 40 liter in vacuoand added to 25 liter ethyl acetate. The ethyl acetate phase wasseparated and concentrated to 1 liter in vacuo. The crude aclacinomycinmixture which precipitated by adding 1 liter of n-hexane to the aboveconcentrate was collected and washed with n-hexane-ethyl acetate mixture(50:1) twice, and 15.5 g. of orange yellow powder was obtained.

This crude powder was dissolved in 200 ml. of ethyl acetate and appliedon a column 35 cm in length and 7 cm in diameter filled with 700 g. ofColumn-Lite and eluted with ethyl acetate-methanol mixture (1:1). Yellowfractions containing aclacinomycin A were evaporated to dryness invacuo. 12.4 g. of crude aclacinomycin A was dissolved in 100 ml. ofchloroform and added to 50 ml. of 10.sup.⁻³ M EDTA in 0.01 M phosphatebuffer (pH 6.8). After shaking vigorously to remove residual metal ions,the chloroform phase was washed twice by shaking with water, dried overanhydrous sodium sulphate and evaporated to dryness in vacuo. 11.1 g. ofaclacinomycin A was obtained as yellow powder.

"Sephadex LH-20" is a lypophilic insoluble molecular-sievechromatographic medium made by cross-linking dextran and marketed byPharmacia, Uppsala, Sweden. The Sephadex LH-20 used in the precedingexamples can be replaced by other similar gel-filtration agents, e.g.Sephadex G25 to G200, Sepharose 4B and 6B (Pharmacia Fine Chemicals AB,Uppsala, Sweden) and Bio-Gel A1.5m (Bio Rad Co.). Preferredgel-filtration agents include the carboxymethyl substituted cross-linkeddextran gels described in columns 3 and 4 of U.S. Pat. No. 3,819,836.

We claim:
 1. A compound of the formula ##SPC2##wherein R represents##SPC3##
 2. The compound of the formula ##SPC4##
 3. The compound of theformula ##SPC5##